PT  - JOURNAL ARTICLE
AU  - Shing, Jiyung
AU  - Jiang, Fuguo
AU  - Liu, Jun-Jie
AU  - Bray, Nicholas L.
AU  - Rauch, Benjamin J.
AU  - Baik, Seung Hyun
AU  - Nogales, Eva
AU  - Bondy-Denomy, Joseph
AU  - Corn, Jacob E.
AU  - Doudna, Jennifer A.
TI  - Disabling Cas9 by an anti-CRISPR DNA mimic
AID  - 10.1101/129627
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 129627
4099  - http://biorxiv.org/content/early/2017/04/23/129627.short
4100  - http://biorxiv.org/content/early/2017/04/23/129627.full
AB  - CRISPR-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known and the potential applications for Cas9 inhibitor proteins in mammalian cells has not fully been established. We show here that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.